ABSTRACT Defects in the biogenesis and function of lysosomes result in lysosomal storage disorders (LSDs). The underlying mechanisms that lead to disease pathology are poorly understood, representing a barrier in identifying therapeutic targets. In the lysosomal disease mucolipidosis II (ML-II), the enzyme (GlcNAc-1- phosphotransferase) that synthesizes the carbohydrate-based tag (mannose 6-phosphate) needed for receptor-mediated lysosomal targeting is missing. This causes hypersecretion of newly made cathepsins into the extracellular space. Cathepsin mistargeting places these enzymes in contact with the matrix-deposited growth factors that control the timing and fidelity of chondrocyte maturation. The primary objective of this proposal is to address how secreted lysosomal cathepsins modulate key growth factor pathways in chondrocytes and cause ML-II cartilage pathology. Most work on ML-II (and other LSDs) focuses on the consequences of storage on cellular function. Our approach is novel in that it focuses instead on the consequences of mistargeted lysosomal hydrolases, a unique feature of ML-II. Using a morpholino- knockdown strategy, we generated the first zebrafish model for ML-II to study the impact of impaired M6P- dependent lysosomal targeting during development. We demonstrated chondrogenesis defects accompanied by sustained expression of the transforming growth factor beta (TGFss)-regulated matrix protein, type II collagen, and sustained and elevated activity of the cathepsin K. Lowering cathepsin K activity in the ML-II background reduced this sustained expression of type II collagen and alleviated the cartilage phenotypes. The finding that inhibition of cathepsin K reduces the abundance of type II collagen was unexpected since decreased activity of this potent collagenase was predicted instead to lead to collagen accumulation. This observation supports a broader function for this protease - beyond collagen turnover - during pathogenic cartilage development. Our collective data support a model whereby ML-II chondrocytes are unable to proceed beyond early phases of chondrogenesis. Since progression through the normal chondrogenic program requires a transition from TGFss to bone morphogenetic protein (BMP) signaling, we hypothesize that hypersecretion and extracellular activity of cathepsin K disrupts the balance of these growth factor pathways, in turn causing abnormal chondrocyte differentiation. ML-II is untreatable. By addressing the molecular mechanisms that underlie abnormal chondrogenesis in ML-II, we hope to identify new downstream targets for therapy and develop a powerful system to investigate the physiological relevance of secreted cathepsins during normal cartilage formation. The proposed specific aims will 1) address how cathepsin K hypersecretion alters the balance of TGFss and BMP signaling during chondrocyte differentiation, 2) define cathepsin K regulation and activity within the stages of normal cartilage development, and 3) uncover the structural elements within the GlcNAc-1-phosphotransferase enzyme that mediate specific recognition of cathepsins.